Single-Molecule Analysis of Hepatitis C Virus NS3 Helicase Translocating on Single-Stranded RNA

Abstract

Large, structured RNA molecules often require the need of RNA helicases in order to correctly and efficiently fold into their active conformation. Likewise, RNA helicases are needed to displace proteins from RNA molecules or to melt RNA structures to make parts of the RNA accessible for binding partners. Long-range translocation events of RNA helicases on their substrates are functionally important, yet lack understanding at the single molecule level. Using the RNA helicase NS3h from Hepatitis C Virus (DExH/D-box family) we used a custom-built “fleezer”, a single-molecule instrument combining fluorescence detection with an optical tweezer, to observe, identify and define underlying translocation events. This instrument provides the necessary means to locate a fluorescence-labeled molecule with the precision of a few nanometers on a long nucleic acid strand that is held under a defined and adjustable tension with piconewton accuracy by optical trapping. We measured long-range translocation of HCV NS3h on single-stranded RNA of various sequences and obtained data on velocity and processivity. With ca. 70 nucleotides per second, NS3h translocation on single stranded RNA is 2-2.5 times faster than on single stranded DNA.